LC-MSN METHOD FOR QUALITATIVE & QUANTITATIVE ANALYSIS OF COMPLEX LIPID MIXTURES

LC-MSN 定性方法

• 批准号: 7955878
• 负责人: Catherine E. Costello
• 金额: $ 0.4万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955878
• 关键词: Ammonium; Biological; Biology; Blood capillaries; Chromatography; Complex; Computer Retrieval of Information on Scientific Projects Database; Detection; Formates; Fractionation; Funding; Genetic; Glycolipids; Grant; Heptanes; High Pressure Liquid Chromatography; Institution; Investigation; Ions; Laboratories; Left; Length; Lipids; Literature; Low-Density Lipoproteins; Mass Spectrum Analysis; Medicine; Methanol; Methodology; Methods; Modification; Molecular; Pattern; Phase; Phospholipids; Plant Resins; Production; Proteins; Publishing; Qualitative Methods; Research; Research Personnel; Resources; Sampling; Scanning; Silicon Dioxide; Source; System; United States National Institutes of Health; Water; base; capillary; feeding; interest; ionization; methyl tert-butyl ether

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While nanospray MS is a good choice for the characterization of simple lipid mixtures (1,2), it is often not sufficient for the qualitative and quantitative analysis of highly complex samples. Most separation methods described are limited, in that they either target only specific classes of interest (3), or are not well suited for MS, the superior detection method, especially for analyses of small amounts of samples. We have developed a simple, reproducible three-step method for lipid analysis by adapting separation systems described in the literature for the chromatography of lipids (4,5). After an optional initial fractionation, normal phase HPLC-MS first provides class separation and then a reversed phase LC-MS/MS system answers remaining questions. Methods: (a) Isolated and extracted LDL lipids and lipid standards are passed stepwise onto and eluted off Silica 60 resin with MTBE (methyl t-butyl ether), followed by methanol. (b) Either these two fractions or the full sample (or set of standards) are further separated on a Waters/YMC microbore PVA-Sil HPLC column and are detected by mass spectrometry in positive and negative ion modes. Two different gradients are used, one based on heptane and MTBE, and one based on MTBE and methanol in the presence of ammonium formate, for the separation of more nonpolar and more polar lipids, respectively. Quantification is based on this step. The former requires a postcolumn feed for proper ionization. (c) Fractions obtained can be further characterized by reversed phase LC-MS/MS using a C18 Atlantis capillary column on a Waters CapLC system interfaced to the triple quadrupole, QoTOF MS, or LTQ-Orbitrap MS, or by nanospray MS/MS and/or precursor ion scanning. Lipid and glycolipid standards containing diverse nonpolar, phospho- and glycolipids have been reproducibly separated on the basis of polarity by elution from Silica 60 resin with MTBE and methanol. This step, when used for biological samples, also serves to protect the following column, but is not always necessary. The sample is separated on a PVA-Sil normal phase column using two different gradients, one for determination of nonpolar lipids, and the other for polar lipids. These separations on the normal phase column allow for an at least semi-quantitative detection. The accuracy of the quantification depends mostly on the quality of internal and external standards available. The collected fractions are partially investigated by nanospray MS (MS/MS, precursor ion scanning and neutral loss scanning) for the determination of the molecular species present. A clean separation of molecular species has been achieved on a reversed phase column. Especially the low abundant PEs can be confirmed that way. The LCMS methodology provides a fairly robust and technically simple method for the investigation of complex lipid mixtures. We have applied the method to the analysis of lipids associated with full-length and truncated apolipiprotein in a normal indivicual and one who has a genetic modification that results in production of the truncated protein. We have found that the lipid pattern is different in the two cases. these results have been published (7) and have drawn significant interest, judging from the number of investigators from the US and elsewhere who have contacted us about this approach as they begin to implement it in their own laboratories. Murphy et al. recently published a modification to the method that simplifies the extraction and chromatography but leaves behind the phospholipids. (8) 1) M. Puffer and R.C. Murphy (2003). Mass Spectrometry Reviews 22, 332-64. 2) X. Han and R.W. Gross (2005). Mass Spectrom Rev. 24, 367-412. 3) R.C. Murphy et al. (2001). Chem. Rev. 101, 479-526. 4) J. Hamilton, and K. Comai (1988). Lipids 23, 1046-49 & 1150-53. 5) W.W. Christie et al. (1995). J. High Resol. Chromatogr. 18, 97-100. 6) F.K. Welty et al. (1991). J. Clin. Invest. 87, 1748-1754. 7) U. Sommer et al. (2006) J. Lipid Res. 47, 804-814. 8) P. M. Hutchins et al. (2008) J. Lipid Res. 49, 804-813.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 虽然nanospray MS是表征简单脂质混合物的良好选择（1，2），但它通常不足以用于高度复杂样品的定性和定量分析。所描述的大多数分离方法都是有限的，因为它们要么仅针对感兴趣的特定类别（3），要么不太适合MS（上级检测方法），特别是对于少量样品的分析。我们已经开发了一种简单的，可重复的三步法脂质分析，采用文献中描述的脂质色谱分离系统（4，5）。在可选的初始分馏后，正相HPLC-MS首先提供类别分离，然后反相LC-MS/MS系统回答剩余的问题。 研究方法：（a）将分离和提取的LDL脂质和脂质标准品逐步传递到硅胶60树脂上并用MTBE（甲基叔丁基醚）洗脱，然后用甲醇洗脱。(b)在沃茨/YMC微孔PVA-Sil HPLC色谱柱上进一步分离这两种馏分或完整样品（或标准品组），并在正离子和负离子模式下通过质谱法进行检测。使用两种不同的梯度，一种基于庚烷和MTBE，一种基于在甲酸铵存在下的MTBE和甲醇，分别用于分离更多的非极性和更多的极性脂质。定量基于此步骤。前者需要柱后进料以进行适当的电离。(c)所获得的级分可通过反相LC-MS/MS（使用连接到三重四极杆的沃茨CapLC系统上的C18 Atlantis毛细管柱）、QoTOF MS或LTQ-Orbitrap MS或通过纳米喷雾MS/MS和/或前体离子扫描来进一步表征。 含有不同非极性、磷脂和糖脂的脂质和糖脂标准品已通过用MTBE和甲醇从硅胶60树脂洗脱而根据极性进行了可重复分离。当用于生物样品时，该步骤也用于保护随后的柱，但并不总是必要的。使用两种不同的梯度在PVA-Sil正相柱上分离样品，一种用于测定非极性脂质，另一种用于测定极性脂质。正相柱上的这些分离允许至少半定量检测。定量的准确性主要取决于可用的内部和外部标准的质量。 通过纳米喷雾MS（MS/MS，前体离子扫描和中性损失扫描）对收集的馏分进行部分研究，以确定存在的分子种类。在反相柱上实现了分子物种的清洁分离。特别是低丰度的PE可以通过这种方式得到确认。 LCMS方法提供了一种相当稳健且技术上简单的方法， 复杂脂质混合物的研究。 我们已经将该方法应用于与正常个体和具有导致产生截短蛋白的遗传修饰的个体中的全长和截短载脂蛋白相关的脂质的分析。我们发现，这两种情况下的脂质模式是不同的。这些结果已经发表（7），并引起了极大的兴趣，从美国和其他地方的研究人员的数量来判断，他们开始在自己的实验室中实施这种方法时与我们联系。Murphy等人最近发表了对该方法的修改，该修改简化了提取和层析，但留下了磷脂。（八） 1)M. Puffer和R.C. Murphy（2003年）。Mass Spectrometry Reviews 22，332 - 64. 2)X. Han和R.W.毛额（2005年）。Mass Spectrom Rev. 24，367 - 412. 3)R.C. Murphy等人（2001年）。101，479 - 526。 4)汉密尔顿和K. Comai（1988年）。Lipids 23，1046 - 49 & 1150 - 53. 5)W.W. Christie等人（1995年）。高解析度J. High Resol. Chromatogr. 18，97 - 100。 6)法光Welty等人（1991年）。J. Clin. Invest. 87，1748 - 1754。 7)联合Sommer等人（2006）J.Lipid Res.47，804 - 814. 8)P. M. Hutchins等人（2008）J.Lipid Res.49，804 - 813.